Abstract Based on mutational studies two decades ago, HIV-1 Integrase (IN) protein has been proposed to play a role in late stages of HIV-1 replication. These mutations, collectively referred to as Class II mutations, adversely affect multiple steps of virus replication including particle assembly, maturation and subsequent reverse transcription. Some class II mutations specifically impair particle maturation leading to the formation of aberrant viral cores in which the viral ribonucleoprotein complexes (RNPs) are mislocalized outside of the conical capsid core. However, for nearly 20 years it has remained enigmatic as to how IN can contribute to proper viral particle maturation. Allosteric IN inhibitors (ALLINIs) have recently emerged as a promising new class of antiretroviral agents and select compounds are currently in clinical trials. Although ALLINIs were initially designed to block the interaction of IN with its cellular cofactor LEDGF/p75, it has recently been shown that they potently impair the late steps of HIV-1 replication. Similar to certain Class II IN mutations, these inhibitors selectively interfere with proper virus particle maturation and yield non-infectious particles with eccentrically positioned RNPs. Although it has been shown that ALLINIs can promote aberrant IN multimerization, how this event adversely results in the mislocalization of the RNPs outside the capsid core has remained unknown. Based on these observations, we have recently explored the intriguing possibility that IN may bind the viral RNA genome in mature particles and that ALLINIs may interfere with IN-RNA interactions critical for proper particle formation. To test this hypothesis, we have employed the cutting-edge CLIP-seq (crosslinking- immunoprecipitation-sequencing) methodology and complementary biochemical approaches. These studies have revealed for the first time that IN binds to specific sequences on the viral RNA genome and that certain mutations within IN and ALLINIs potently block these interactions. We propose to continue our studies in identifying the details of the mechanism by which ALLINIs affect IN-RNA interactions and how IN-RNA interactions regulate particle maturation. We also propose to use the aberrantly formed eccentric cores generated in the presence of ALLINIs/Class II mutations as a tool to understand the early post-entry events in infection. As such, this project will not only provide unprecedented insight into the novel role of HIV-1 IN in particle assembly and the primary mechanism of action of ALLINIs, but will also facilitate the development of studies to understand early post-entry events in HIV-1 replication.